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RT-PCR procedures

Advantages of Two-step RT-PCR Procedure. A two-step procedure has the following advantages:
  • Optimizes reaction conditions. The two-step format allows both reverse transcription and PCR to be performed under optimal conditions to ensure efficient and accurate amplification.
  • Provides flexibility. Two-step procedures allow the product of a single cDNA synthesis reaction to be used for analysis of multiple transcripts. This flexibility is valuable for such specialized applications as rapid amplification of cDNA ends (RACE) and Differential Display Reverse Transcription (DDRT).
  • Amplifies long sequences. With the right combination of reverse transcriptase and thermostable DNA polymerase, two-step RT-PCR can amplify RNA sequences up to 14 kb long.
2. One tube, one-step procedure (coupled RT-PCR) Both cDNA synthesis and PCR amplification are performed with the same buffer and site-specific primers, eliminating the need to open the reaction tube between the RT and PCR steps. In addition to the higher sensitivity of this approach (as in the one tube, two-step reaction above), the one-step approach minimizes the chance of contamination, since the entire reaction is performed with minimal pipetting steps and without opening the tube. In addition, this approach permits direct analysis of a specific transcript, since the primers used in both steps are sequence-specific. Finally, the thermoactive reverse transcriptase used in this procedure allow a high RT
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